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1.
P.R. Andjus M.R. Djurišić Z. Žujović N. Begović R. Srejić D. Vučelić 《The Journal of membrane biology》1999,167(3):267-274
The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability
of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P
d
) were observed delimiting a region of low activation energy (E
a
around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region
water transport was of a different type with high E
a
(about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at
15–30°C and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl− channel inhibitor, ethacrynic acid, diminished P
d
in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate)
the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased P
d
below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence
of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining
membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in
the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at
13 and 29°C.
Received: 12 August 1998/Revised: 13 November 1998 相似文献
2.
Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E50, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal
activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant
of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105
mm K+, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C.
In Na+-rich solution with 2.5 mm extracellular K+γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K+ over Na+. γ depended on extracellular K+ concentration (K
D
= 19.6 mm) and temperature (Q
10= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested
with a half-maximal inhibiting concentration (IC50) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX,
IC50= 6.8 nm) and mast cell degranulating peptide (MCDP, IC50= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and
DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane
potential.
Received: 2 June 1995/Revised: 13 October 1995 相似文献
3.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
4.
cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative
PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected
cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering
was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting
an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding
potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current
amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35
mV (half activation, V
0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and
24.6 mV (slope factor) and −17.3 and −39.0 mV (V
0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials
ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at
−30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels
were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the
addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively.
8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel.
Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels
to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel
was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time
constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2
subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics
when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
Received: 18 June 1997/Revised: 7 October 1997 相似文献
5.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels
had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+
o
//155 mm K+
i
) and 231 ± 4 pS in external high-K
o
solution (155 mm K+
o
//155 mm K+
i
). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3
m internal Ca2+, respectively. Using an internal solution containing 10−4
m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with
a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones.
Received: 7 November 1996/Revised: 29 October 1997 相似文献
6.
We used 31P NMR to investigate the temperature-dependence of intracellular pH (pH
i
) in isolated frog skeletal muscles. We found that ln[H+
i
] is a linear function of 1/T
abs paralleling those of neutral water (i.e., H+= OH−) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected
by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout
resulted in an amiloride-sensitive return to the (temperature dependent) basal pH
i
.
These results show that the temperature dependence of activation of Na+-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/[OH−] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point
of the Na+-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated
conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation
of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation,
suggesting that changes in Arrhenius activation energy (E
a
) may not be a part of the mechanism of hormone stimulation.
Received: 12 February 1997/Revised: 1 October 1997 相似文献
7.
Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity
cellobiose buffer. The osmotic water permeability coefficient P
f
for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10−3 cm sec−1 at 23°C, a value lower than 3.6 × 10−3 cm sec−1 exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E
a
of 14.7 Kcal mol−1 for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol−1 determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E
a
, as well as the low P
f
for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with
the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the
osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could
not be observed in the eel intestinal vesicles. The P
f
dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure
and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume
transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in
kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of
swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10−3 N cm−1, compares favorably with the corresponding value, 0.87 × 10−3 N cm−1, estimated from measurements at osmotic equilibrium.
Received: 28 January 1999/Revised: 15 June 1999 相似文献
8.
Pyrethroid modulation of sodium channels is unique in the sense that it is highly dependent on temperature, the potency being
augmented by lowering the temperature. To elucidate the mechanisms underlying the negative temperature dependence of pyrethroid
action, single sodium channel currents were recorded from cultured rat hippocampal neurons using the inside-out configuration
of patch-clamp technique, and the effects of the pyrethroid tetramethrin were compared at 22 and 12°C. Tetramethrin-modified
sodium channels opened with short closures and/or transitions to subconductance levels at 22 and 12°C. The time constants
of the burst length histograms for tetramethrin-modified channels upon depolarization to −60 mV were 7.69 and 14.46 msec at
22 and 12°C, respectively (Q10= 0.53). Tetramethrin at 10 μm modified 17 and 23% of channels at 22 and 12°C, respectively, indicating that the sensitivity of the sodium channel of rat
hippocampal neurons to tetramethrin was almost the same as that of tetrodotoxin-sensitive sodium channels of rat dorsal root
ganglion neurons and rat cerebellar Purkinje neurons. The time constants for burst length in tetramethrin-modified sodium
channels upon repolarization to −100 mV from −30 mV were 8.26 and 68.80 msec at 22 and 12°C (Q10= 0.12), respectively. The prolongation of tetramethrin-modified whole-cell sodium tail currents upon repolarization at lower
temperature was ascribed to a prolongation of opening of each channel. Simple state models were introduced to interpret behaviors
of tetramethrin-modified sodium channels. The Q10 values for transition rate constants upon repolarization were extremely large, indicating that temperature had a profound
effect on tetramethrin-modified sodium channels.
Received: 31 January 2000/Revised: 18 May 2000 相似文献
9.
The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1–30 mm K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K+ activated current through outward-rectifying K+ channels (I
K,
out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]
o
shifted the voltage-sensitivity of I
K,
out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]
o
was evident in the kinetics of I
K,
out activation and deactivation, as well as the steady-state conductance- (g
K
−) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K+, −80 mV in 1.0 mm K+, and −20 mV in 10 mm K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+≥ Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g
K
or on I
K,
out activation kinetics, although 10 mm [Ca2+]
o
accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K+]
o
suppressed g
K
completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of I
K,
out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the
channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of
two K+ ions outside affects the distribution between closed states of the channel.
Received: 27 November 1996/Revised: 4 March 1997 相似文献
10.
A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis
The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode
and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells' (Wang,
J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma
196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species.
Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl− channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K+ channel (VKC1, Valonia K
+
Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl− media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0
± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl− and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl− media (bath/pipette; E
Cl−
−20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl−, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann
fits of the channel activity under symmetrical 150 mm Cl− conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl− gradient.
VAC2 was activated by voltages more negative than E
Cl−
and was always observed together with VAC1, but less frequently. It showed a ``flickering' gating. The single channel conductance
was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel
conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance
state had a single channel conductance of 124 ± 27 pS (n= 6).
In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl− conductance of the plasmalemma of the turgescent ``mother cells' and that this channel (as well as VAC2) can operate in
the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related
(as the stretch-activated channels) to processes involved in turgor regulation.
Received: 24 June 1999/Revised: 2 September 1999 相似文献
11.
H. Chabot M.F. Vives A. Dagenais Cz. Grygorczyk Y. Berthiaume R. Grygorczyk 《The Journal of membrane biology》1999,169(3):175-188
Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel
(ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR
depends on the ability of CFTR to conduct Cl− into the cell and is negligible when Cl− flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (I
amil
) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with
1 mm IBMX reduced I
amil
by up to 80%, demonstrating that ENaC is inhibited when Cl− is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I
amil
downregulation and the CFTR-mediated plasma membrane Cl− conductance, suggesting a direct correlation. However, I
amil
downregulation was not affected when Cl− flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl− reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I
amil
downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in I
amil
downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit I
amil
. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve
Ca2+ and is independent of the direction and magnitude of Cl− transport across the plasma membrane.
Received: 15 December 1998/Revised: 5 March 1999 相似文献
12.
A. Chorvatova A. Guyot C. Ojeda O. Rougier A. Bilbaut 《The Journal of membrane biology》1998,162(1):39-50
The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the
whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which
began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These
effects were abolished by Losartan (10−5
m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized
in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method,
a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal
stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau
current. In Cl−-free solution, the outward current reversed at −78.5 mV, a value close to E
K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K+-free solution, the transient outward current, sensitive to Cl− channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E
Cl. Its magnitude changed in the same direction as the driving force for Cl−. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca2+-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin
II is due to Ca2+-dependent K+ and Cl− currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]
i
originating from intra- and extracellular stores.
Received: 29 May 1997/Revised: 4 November 1997 相似文献
13.
The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl− absorption linked to Na+ were investigated in isolated perfused tubules using Cl−-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl− (a
i
Cl
) is 11.3 ± 0.5 mm, the basolateral (V
1
), apical (V
2
), and transepithelial (V
3
) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, a
i
Cl
falls by 1.3 ± 0.3 mm and V
2
hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na+ from the lumen causes a
i
Cl
to fall further by 2.3 ± 0.4 mm and V
2
to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a
i
Cl
was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide
or hydrochlorothiazide have no effect on a
i
Cl
. The effects of luminal HCO−
3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO−
3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven
Cl-HCO3 exchange. Several lines of evidence favor conductive Cl− transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate
to the lumen or bath, increases the a
i
Cl
by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO−
3-free Ringer, the equilibrium potential for Cl− does not differ from the membrane potential V
2
. Finally, the logarithmic changes in a
i
Cl
in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane
potential. These findings strongly support the presence of Cl− channels at the apical and basolateral cell membranes of the proximal tubule.
Received: 14 November 1997/Revised: 6 July 1998 相似文献
14.
We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes
of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with
a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl− than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with
an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could
also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease
of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis
of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern
was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability
sequence for test halides determined from reversal potentials was Br− > Cl− > I−≈ F−. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br− and Cl− have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel
activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl−. Our results suggest that channel block involves the interaction of HPO2−
4 with a site located at 70% of the membrane span.
Received: 10 January 1997/Revised: 29 May 1997 相似文献
15.
Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation 总被引:3,自引:0,他引:3
Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia
cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report
that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control
cells. The membrane potential was repolarized by increased extracellular K+ concentration (56 mV per 10-fold change in K+ concentration). Using the cell-attached patch-clamp technique, K+ channel activity was 1.7 times higher in mcl-1 transfected cells (NP
o
= 22.7 ± 3.3%) than control cells (NP
o
= 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively.
Suppression of K+ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K+ channel activity.
Received: 30 March 1999/Revised: 20 July 1999 相似文献
16.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
17.
The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites
for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation—through effects on
membrane potential, Ca2+ signalling, and interleukin-2 secretion—and in cytotoxic killing and volume regulation. Because PKC activation (as well as
increased intracellular Ca2+) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by
+8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action
with ``death brew' (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K+ conductance (G
K
) by 41 ± 2%. PKC activation by 4β-phorbol 12,13-dibutyrate, increased G
K
by 69 ± 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase
in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate
or substrate peptides reduced G
K
by 43 ± 5% and 38 ± 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose-
and light-dependent manner (IC50∼ 250 nm). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts
in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased
Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca2+ entry in the presence of activating stimuli.
Received: 12 July 1996/Revised: 21 October 1996 相似文献
18.
We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH2-terminal domain of the Shaker K+ channel with purified, ryanodine-modified, cardiac Ca2+-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a
holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a
single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec−1μm
−1 for ShB and 2000 ± 200 sec−1μm
−1 for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied
potential with the bulk of the voltage-dependence residing in Koff. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As
is the case with inactivation and block of K+ channels, this is mediated by a large increase in Kon. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites
which will contribute to the ion handling properties of this channel.
Received: 15 December 1997/Revised: 13 March 1998 相似文献
19.
Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either
standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated
method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using
the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm
of [K+]
e
with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n= 4. Time constants ranged from 1 to 4 msec with a maximum at −25 mV. The steady-state activation curve spanned the voltage
range −50 to +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time
constants τ1 and τ2 were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation
was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage
range −40 to −20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of
this ``very slow inactivation' process (τvs) determined for voltage steps at −60 and −50 mV were 15 and 10 sec respectively. A ``facilitation process' of the peak current
was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range −100 to −50 mV.
Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In
some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed
in relation with the membrane electrogenesis of this cell type.
Received: 14 November 1994/Revised: 24 October 1995 相似文献
20.
A.E. Alekseev M.E. Kennedy B. Navarro A. Terzic 《The Journal of membrane biology》1997,159(2):161-168
Co-expression of clones encoding Kir6.2, a K+ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K+ (KATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1
channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 ± 0.1 to 0.4 ± 0.03 msec,
and from 0.4 ± 0.1 to 2.0 ± 0.2 msec, and mean open times from 1.9 ± 0.4 to 4.5 ± 0.8 msec, and from 12.1 ± 2.4 to 5.0 ± 0.2
msec between −100 and −20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17.9 ± 1.8 msec
with 5.6 ± 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between
one open and three closed states with forward and backward rate constants of 1905 ± 77 and 322 ± 27 sec−1 for intraburst, 61.8 ± 6.6 and 23.9 ± 5.8 sec−1 for interburst, 12.4 ± 6.0 and 13.6 ± 2.9 sec−1 for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable
from pancreatic or cardiac KATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native KATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac KATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic
identity of pancreatic, but not cardiac KATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization
of novel KATP channel clones.
Received: 12 March 1997/Revised: 5 May 1997 相似文献